The gene product of XRCC4 has been implicated in both V(D)J recombination and the more general process of double strand break repair (DSBR). To date its role in these processes is unknown. Here, we describe biochemical characteristics of the murine XRCC4 protein. XRCC4 expressed in insect cells exists primarily as a disulfide-linked homodimer, although it can also form large multimers. Recombinant XRCC4 is phosphorylated during expression in insect cells. XRCC4 phosphorylation in Sf9 cells occurs on serine, threonine, and tyrosine residues.We also investigated whether XRCC4 interacts with the other factor known to be requisite for both V(D)J recombination and DSBR, the DNA-dependent protein kinase. We report that XRCC4 is an efficient in vitro substrate of DNA-PK and another unidentified serine/ threonine protein kinase(s). Both DNA-PK dependent and independent phosphorylation of XRCC4 in vitro occurs only on serine and threonine residues within the COOH-terminal 130 amino acids, a region of the molecule that is not absolutely required for XRCC4's DSBR function. Finally, recombinant XRCC4 facilitates Ku binding to DNA, promoting assembly of DNA-PK and complexing with DNA-PK bound to DNA. These data are consistent with the hypothesis that XRCC4 functions as an alignment factor in the DNA-PK complex.V(D)J recombination is the process of assembling the variable (V), diversity (D), and joining (J) gene segments of the immunoglobulin and T cell receptor variable region genes during development of B and T lymphocytes (1, 2). The proteins responsible for V(D)J joining are collectively referred to as the V(D)J recombinase, and are shared by both the B and T cell lineages. It has recently become clear that the lymphocytespecific proteins, recombination activating genes 1 and 2 (RAG 1 1 and RAG 2) (3, 4) are directly responsible for initiation of the V(D)J recombination reaction (5-8) together directing cleavage at the recombination signal sequence-coding juncture; whereas resolution of recombination intermediates also requires several ubiquitously expressed DNA repair factors. Evidence linking DNA repair activities with V(D)J recombination arose from the characterization of cells from homozygous severe combined immune deficient (SCID) mice (9 -11). C.B-17 SCID mice which are immunodeficient because of defective V(D)J recombination, were shown to also be defective in repairing DSBs in lymphoid and non-lymphoid cells (12, 13). Further evidence for an overlap in the activities of V(D)J recombination and DSB repair came from the observation that radiosensitive rodent cell lines which were defective in DSB repair could not support V(D)J recombination induced by co-transfecting RAG 1 and RAG 2; whereas, radiosensitive mutants proficient in DSB rejoining were normal in this capacity (14 -16). Four factors have been delineated which are required for both V(D)J recombination and DSBR; three of these encode components of the DNA-dependent protein kinase (17-25). DNA-PK is a nuclear serine/ threonine protein kinase whose catalytic ...

V(D)J rearrangement is the molecular mechanism by which an almost infinite array of specific immune receptors are generated. Defects in this process result in profound immunodeficiency as is the case in the C.B-17 SCID mouse or in RAG-1 (recombination-activating gene 1) or RAG-2 deficient mice. It has recently become clear that the V(D)J recombinase most likely consists of both lymphoidspecific factors and ubiquitously expressed components of the DNA double-strand break repair pathway. The deficit in SCID mice is in a factor that is required for both of these pathways. In this report, we show that the factor defective in the autosomal recessive severe combined immunodeficiency of Arabian foals is required for (i) V(D)J recombination, (ii) resistance to ionizing radiation, and (iii) DNA-dependent protein kinase activity.During early lymphoid differentiation distinct gene segments called variable (V), diversity (D), and joining (J) are joined to form the coding sequences of immunoglobulin and T-cell antigen receptor variable regions. This process depends upon site-specific somatic recombination and results in the random assortment of various combinations of V, D, and J gene segments (reviewed in refs. 1-3). The rearrangement process involves two double-stranded DNA cuts and subsequent religations. This results in the formation of two new DNA joints-coding joints, which contain the coding information, and signal joints, which contain the two recombination signal sequences (ref. 3 and references therein). In 1989 and 1990, two highly conserved genes were discovered, RAG-1 and RAG-2 (recombination-activating genes 1 and 2), which are clearly essential for V(D)J recombinase activity (4, 5) and have recently been directly implicated in initiation of V(D)J recombination (6).In 1983, Bosma et al. (7) described a spontaneous mutation in C.B-17 mice which phenotypically resembled a human lymphoid deficiency disease, severe combined immunodeficiency (SCID). In 1986, it became clear that the deficiency in C.B-17 mice is in the step of coding-joint ligation of V(D)J recombination (8-10). In 1990 it was reported that the mutation in SCID mice not only affects V(D)J recombination but also impairs the more general process of double-strand break repair (DSBR) (11, 12). Thus, this was the first suggestion that the V(D)J recombinase might utilize ubiquitous DNA repair factors to carry out the V(D)J recombination reaction.DSBR is vital to all organisms because it ensures integrity of chromosomes. Studies with mammalian cells that are hypersensitive to agents that induce chromosomal breaks have defined three distinct complementation groups that are deficient both in rejoining double-strand breaks and in their ability to support RAG-induced V(D)J recombination (xrs, XR-1,The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. and murine SCID; refs. 13 and 14). Thus, the eme...

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